Albumin is the most abundant plasma protein, generally constituting slightly over one-half of the total protein in mammalian plasma. In the human body, albumin has the important role of regulating the water balance between blood and tissues, and of functioning as a transport molecule for various compounds, such as bilirubin, fatty acids, cortisol, thyroxine and drugs such as sulfonamides and barbiturates, that are only slightly soluble in water. An albumin deficiency can restrict the transport of slightly water soluble materials throughout the body and a deficiency is signaled in an individual by an abnormal accumulation of serous fluid, or edema. Therefore, it is clinically important to determine whether an individual has a deficiency of serum albumin.
Likewise, it is clinically important to determine if an individual is excreting an excess amount of protein. A normal functioning kidney forms urine i essentially a two step process. Blood flows through the glomerulus, or glomerular region of the kidney. The capillary walls of the glomerulus are highly permeable to water and low molecular weight components of the blood plasma. Albumin and other high molecular weight proteins cannot pass through these capillary walls and are essentially filtered out of the urine so that the protein is available for use by the body. The liquid containing the low molecular weight components passes into the tubules, or tubular region, of the kidney where reabsorption of some urine components, such as low molecular weight proteins; secretion of other urine components; and concentration of the urine occurs. As a result, through the combined processes of the glomerulus and tubules, the concentration of proteins in urine should be minimal to absent. Therefore, abnormally high amounts of albumin or other proteins in urine must be detected and related to a physiological dysfunction.
The relatively high concentration of albumin in the urine of an individual usually is indicative of a diseased condition. For example, the average normal concentration of protein in urine varies from about 2 mg/dL to about 8 mg/dL, with approximately one-third of the total urinary protein being serum albumin. However, in a majority of diseased states, urinary protein levels increase appreciably, such that albumin accounts for from about 60 percent to about 90 percent of the excreted protein. The presence of an abnormal increased amount of protein in the urine, known as proteinuria, is one of the most significant indicators of renal disease, and may be indicative of various other non-renal related diseases.
Therefore, in order to determine if an individual either has an albumin deficiency or excretes an excess amount of protein, and in order to monitor the course of medical treatment to determine the effectiveness of the treatment, simple, accurate and inexpensive protein detection assays have been developed Furthermore, of the several different assay methods developed for the detection or measurement of protein in urine and serum, the methods based on dye binding techniques have proven especially useful because dye binding methods are readily automated and provide reproducible and accurate results.
In general, dye binding techniques utilize pH indicator dyes that are capable of interacting with a protein, such as albumin, and that are capable of changing color upon interaction with a protein absent any change in pH. When a pH indicator dye interacts with, or binds to, a protein, the apparent pK.sub.a (acid dissociation constant) of the indicator dye is altered and the dye undergoes a color transition, producing the so-called "protein-error" phenomenon. In methods utilizing the dye binding technique, an appropriate buffer maintains the pH indicator dye at a constant pH to prevent a color transition of the pH indicator dye due to a substantial shift in pH. Due to the "protein-error" phenomena, upon interaction with the protein, the pH indicator dye undergoes a color transition that is identical to the color change arising because of a change in the pH. Examples of pH indicator dyes used in the dry phase assay of proteins that are capable of interacting with or binding to proteins and exhibiting "protein-error" color transitions include tetrabromophenol blue and tetrachlorophenol-3,4,5,6-tetrabromosulfophthalein.
Although pH indicator dyes have been used extensively in protein assays, several disadvantages still exist in protein assay methods utilizing indicator dyes. For example, methods based upon pH indicator dyes either cannot detect or cannot quantitatively differentiate between protein concentrations below approximately 15 mg/dL. In addition, although several simple semiquantitative tests and several complex quantitative tests are available for the determination of the total protein content in a test sample, the majority of these assay methods, with the notable exception of the simple colorimetric reagent test strip, require the precipitation of protein to make quantitative protein determinations.
The colorimetric reagent test strip utilizes the previously discusses ability of proteins to interact with certain acid-base indicators and to alter the color of the indicator without any change in the pH. For example, when the indicator tetrabromophenol blue is buffered to maintain a constant pH of approximately 3, the indicator imparts a yellow color to solutions that do not contain protein. However, for solutions containing protein, the presence of protein causes the buffered dye to impart either a green color or a blue color to solution, depending upon the concentration of protein in the solution.
Some colorimetric test strips used in protein assays have a single test area consisting of a small square pad of a carrier matrix impregnated with a buffered pH indicator dye, such as tetrabromophenol blue. Other colorimetric test strips are multideterminant reagent strips that include one test area, or test pad, for protein assay as described above, and further include several additional test pads on the same strip to permit the simultaneous assay of other urinary constituents. For both types of colorimetric test strips, the assay for protein in urine is performed simply by dipping the colorimetric test strip into a well mixed, uncentrifuged urine sample, then comparing the resulting color of the test pad of the test strip to a standardized color chart provided on the colorimetric test strip bottle.
For test strips utilizing tetrabromophenol blue, buffered at pH 3, as the indicator dye, semiquantitative assays for protein can be performed and are reported as negative, trace, or one "plus" to four "plus". A negative reading, or yellow color, indicates that the urine contains no protein, as demonstrated by the lack of a color transition of the indicator dye. A trace reading may indicate from about 5 to 20 mg/dL of protein in the urine. The one "plus" to four "plus" readings, signified by color transitions of green through increasingly dark shades of blue, are approximately equivalent to urine protein concentrations of 30, 100, 300, and over 2000 mg/dL, respectively, and serve as reliable indicators of increasingly severe proteinuria.
In accordance with the above-described method, an individual can readily determine, visually, that the protein content of a urine sample is in the range of 0 mg/dL to about 30 mg/dL. However, the color differentiation afforded by the presently available commercial test strips is insufficient to allow an accurate determination of protein content in urine between 0 mg/dL and about 15 mg/dL. The inability to detect and differentiate between low protein concentrations is important clinically because a healthy individual usually has a urine protein level in the range of about 10 mg/dL to about 20 mg/dL. Therefore, it could be clinically important to know more precisely the urine protein content of an individual, rather than merely estimating the protein content at some value less than about 30 mg/dL.
Of course, the protein content of a urine sample can be determined more precisely by semiquantitative protein precipitation techniques or by quantitative 24 hour protein precipitation techniques. However, these tests are time consuming and relatively expensive. Furthermore, the precipitation tests must be run in a laboratory by trained personnel, and therefore are unavailable for the patient to perform at home to quickly determine urine protein content and to monitor the success or failure of a particular medical treatment.
Therefore, it would be extremely advantageous to have a simple, accurate and trustworthy method of assaying urine for protein content that allows visual differentiation of protein levels in the ranges of 0 mg/dL to about 5 mg/dL, about 5 mg/dL to about 10 mg/dL, and about 10 mg/dL to about 15 mg/dL, and upwards to between about 30 mg/dL to about 300 mg/dL. By providing such an accurate method of determining urine protein concentration in an easy to use form, like a dip-and-read test strip, the urine assay can be performed by laboratory personnel to afford immediate test results, such that a diagnosis can be made without having to wait up to one day for assay results and medical treatment can be commenced immediately. In addition, the test strip method can be performed by the patient at home to more precisely monitor low levels of protein in urine and/or the success of the medical treatment the patient is undergoing. Finally, the method and test device used in a protein assay should not adversely affect or interfere with other test pads that are present on a multi-determinant test strip.
As will be described more fully hereinafter, the method of the present invention allows the fast, accurate and trustworthy protein assay of urine by utilizing a test strip that includes a test pad comprising a new and improved carrier matrix incorporating an indicator reagent composition. The new and improved carrier matrix comprises a film, membrane or layer of a polymerized urethane-based compound that, surprisingly and unexpectedly, substantially improves the sensitivity and accuracy of protein assays by enhancing the color resolution and color differentiation of the assay. Accordingly, urine protein concentrations can be determined accurately at levels as low as about 5 mg/dL. Therefore, in general, the carrier matrix of the present invention provides an improved color resolution of the color transition resulting from contact of the protein-containing test sample with the indicator reagent composition. Consequently, assay sensitivity is improved, and the detection and measurement of protein content in liquids at levels as low as about 5 mg/dL is achieved.
Macroproteinuria or microproteinuria resulting either from abnormally high or abnormally low albumin levels depends upon the precise nature of the clinical and pathological disorder and upon the severity of the specific disease. Proteinuria can be intermittent or continuous, with transient, intermittent proteinuria usually being caused by physiologic or functional conditions rather than by renal disorders. Therefore, accurate assays of urine and other test samples for protein must be available for both laboratory and home use. The assays must permit the detection or measurement of proteins such that a correct diagnosis can be made and correct medical treatment implemented, monitored and maintained. In addition, it would be advantageous if the protein assay method, either for high concentrations of proteins or for low concentrations of proteins, is in a dip-and-read format for the easy and economical, qualitative or quantitative determination of protein in urine or other test samples.
Furthermore, any method of assaying for protein in urine or other test samples must yield accurate, trustworthy and reproducible results by utilizing a method that provides a detectable or measurable color transition as a result of an interaction between the indicator reagent composition and the protein, and not as a result of a competing chemical or physical interaction, such as a pH change or preferential interaction with a test sample component other than protein. Moreover, it would be advantageous if the protein assay method is suitable for use in dry reagent strips for the rapid, economical and accurate determination of protein in urine and other test samples. Additionally, the method and test pad, comprising the carrier matrix and the indicator reagent composition, utilized in the assay for protein should not adversely affect or interfere with the other test reagent pads that are present on multideterminant test strips.
Prior to the present invention, no known method of assaying urine or other test samples for proteins utilized a test device including a test pad comprising an indicator reagent composition homogeneously incorporated into a carrier matrix comprising a film, membrane or layer of a polymerized urethane-based compound. The new carrier matrix provides improved color resolution and increased assay sensitivity compared to present day carrier matrices, thereby achieving accurate and trustworthy protein assays for protein concentrations as low as about 5 mg/dL.
In addition, although a dry phase chemistry test strip utilizing a dye, such as tetrabromophenol blue or tetrachlorophenol-3,4,5,6-tetrabromosulfonephthalein, has been used extensively for several years, no dry phase test strip has utilized a test pad comprising a film, membrane or layer of a polymerized urethane-based compound. The carrier matrix improves color resolution and increases assay sensitivity, especially at lower protein concentration levels, such as protein levels of about 15 mg/dL and less. Furthermore, until the method of the present invention, dry phase test strip procedures were available principally to test for total protein concentration, i.e., for albumin, only down to levels as low as about 30 mg/dL. However, surprisingly and unexpectedly, because of the increased assay sensitivity afforded by the new and improved carrier matrix, the method of the present invention provides a dry phase test strip assay of urine and other test samples for protein down to levels as low as about 5 mg/dL.
The prior art contains numerous references on the wet phase and the dry phase chemistry utilized in the pH indicator dye method of assaying urine for proteins. For example, Keston U.S. Pat. No. 3,485,587 discloses the basic dye binding technique used to assay for proteins at a constant pH. Keston teaches utilizing a single indicator dye, maintained at a constant pH slightly below the pK.sub.a (acid dissociation constant) of the dye and impregnated into a dry test paper, like filter paper, to determine the presence or concentration of albumin by monitoring the color transition of the dye. Free, et al., in U.S. Pat. No. 3,095,277, also discloses a method of detecting the albumin content of liquid test samples by incorporating a suitable indicator composition into a bibulous carrier matrix, like untreated filter paper. Similarly, Atkinson et al. in U.S. Pat. No. 3,438,737 discloses a test device comprising a test composition impregnated into an untreated bibulous matrix, such as filter paper, wood strips, synthetic plastic fibrous materials, non-woven fabrics and woven fabrics for detecting protein in fluids.
Japanese Patent No. 60-49256 is directed to a water phase protein assay utilizing an indicator composition including Coomassie Brilliant Blue dye, methylcellulose, and an acid having a pK.sub.a of from zero to four. The wet phase assay for proteins utilizing Coomassie Brilliant Blue dye also is described in the publication by M. M. Bradford, "A Rapid and Sensitive Method for the Quantitation of Microgram Quantities of Protein Utilizing the Principle of Protein Dye Binding", Anal.Bio. 72, 248-256 (1976). However, although this wet phase assay is sensitive to low protein concentrations, the wet phase assay also is impractical and cumbersome compared to a dry phase assay. For example, the Coomassie Brilliant Blue dye leads to excessive staining of glassware and other assay apparatus. In contrast, a dry phase test strip is discarded after use thereby avoiding costly and time-consuming manipulative steps, such as cleaning the stained glassware and other assay apparatus.
Smith-Lewis et al., in U.S. Pat. No. 4,166,093, disclosed a multi-layered dry phase test device including a layer comprising a polymer and, optionally, a finely-divided particulate material. This polymer-based layer is included in the test device to reflect, or absorb, detecting radiation and thereby facilitate detection of the analyte of interest. Wu et al. in U.S. Pat. No. 4,274,832 disclosed a similar radiation-blocking layer including either an opacifying agent, such as an inorganic metal salt, like titanium dioxide, or a non-fibrous, film-forming natural or synthetic polymer, like gelatin or a polyvinyl compound, or combinations thereof.
Siddiqi, in U.S. Pat. No. 4,438,067, disclosed a dry phase test device wherein distinct polymeric beads, incorporating the indicator reagent, were applied to a nonporous base, such as a plastic or a metal. The polymeric beads comprise a water-insoluble hydrophilic polymer, like cellulose and hydroxyacrylic polymers. The color transition resulting from contact of the test device with a test sample occurs within the beads. According to the method of Siddiqi, the indicator reagent is incorporated into the polymeric beads before the beads are applied to the support of the test device.
Tanny U.S. Pat. No. 4,466,931, described a method of manufacturing a permeable membrane by rapidly polymerizing a thin layer of a solution of a monomer or an oligomer to form an insoluble polymer. The solvent of the solution then is removed to provide a microporous membrane. Tanny disclosed a rapid polymerization of monomers or oligomers by ultraviolet or electron beam radiation to form a microporous membrane. Ford, in U.S. Pat. No. 4,661,526, disclosed a method of preparing a crosslinked, porous polymeric membrane formed from polyamides or polyamide/polyimide copolymers.
However, none of the above-cited references teaches or suggests either alone or in combination, that a carrier matrix, comprising a film, membrane or layer of a polymerized urethane-based compound, can be used in a diagnostic device to achieve a more accurate determination of the amount of an analyte, like protein, and especially low amounts of an analyte, like about 4 mg/dL, in a test sample. In contrast to the prior art, and in contrast to the presently available commercial test strips, the method of the present invention provides increased accuracy and increased sensitivity in the detection and measurement of proteins in a liquid test sample, such as a biological fluid, like urine. Surprisingly and unexpectedly, by utilizing a carrier matrix of the present invention, protein levels of about 30 mg/dL and below, down to about 5 mg/dL, can be determined accurately. Hence, in accordance with the method of the present invention, new and unexpected results are achieved in the dry phase reagent strip assay of urine and other test samples for proteins by utilizing a test pad including an indicator reagent composition incorporated into a carrier matrix comprising a film, membrane or layer of a polymerized urethane-based compound.